Neutrophils are the most abundant cell type among circulating white blood cells and constitute the first line of host defense against invading bacteria and other pathogens. They are terminally differentiated cells and normally have a very short life-span (6-7 hours in blood and 1-4 days in tissue). Neutrophils readily undergo spontaneous programmed cell death (apoptosis) and this death program needs to be well controlled to maintain the normal neutrophil count. Accelerated neutrophil death leads to a decrease of neutrophil counts (neutropenia), while delayed neutrophil death elevates neutrophil counts (neutrophilia). Delayed clearance of neutrophils in tissues causes unwanted and exaggerated inflammation. The long-term goal of this project is to elucidate the molecular basis of this finely regulated neutrophil spontaneous death. The susceptibility of cells to apoptosis appears to be dependent on the balance between pro-apoptotic and pro-survival (anti-apoptotic) signals. We recently established deactivation of Ptdlns(3,4,5)P3/Akt, a well known cellular survival signal, as one of the causal mediators of apoptosis in a variety of cells. We also investigated the involvement of Ptdlns(3,4,5)P3/Akt signaling in neutrophil spontaneous death. Our preliminary data show that Ptdlns(3,4,5)P3/Akt signal is dramatically deactivated during neutrophil death. Inhibition of this signal pathway promotes neutrophil spontaneous death, while augmentation of this signal prevents neutrophil death. Accordingly, we hypothesize that Ptdlns(3,4,5)P3/Akt deactivation acts as a physiological mediator in neutrophil spontaneous death. To further understand the involvement of Ptdlns(3,4,5)P3/Akt signaling in neutrophil death, we will characterize the molecular mechanisms by which Ptdlns(3,4,5)P3/Akt activity is down regulated during neutrophil spontaneous death (Aim I). Moreover, the downstream mechanisms responsible for Ptdlns(3,4,5)P3/Akt deactivation-mediated neutrophil death will be investigated (Aim II). Finally, the contribution of Ptdlns(3,4,5)P3/Akt pathway to neutrophil death in live animals will be investigated using a mouse peritonitis model (Aim III). Together, these studies will provide a better understanding of the role of Ptdlns(3,4,5)P3/Akt in neutrophil spontaneous death, with the ultimate goal of establishing Ptdlns(3,4,5)P3/Akt and related pathways as therapeutic targets for modulating neutrophil functions. Thus, more efficient and effective therapies could be developed to treat a variety of infectious and inflammatory diseases. In addition, defining the molecular basis of neutrophil spontaneous death will assist us to design novel clinical procedures for the long-term storage and application of neutrophils in transfusion medicine. [unreadable] [unreadable] [unreadable]